The pregnant woman faces a unique physiological challenge to reorganize the maternal uterine vascular network to accommodate the metabolic demands of the fetoplacental unit. At the same time and in a coordinated fashion the placenta itself must establish villous circulation for delivery of maternal nutrients to the growing fetus. In this program project we propose to extend our understanding of basic mechanisms and pathophysiological alterations that regulate vasodilatation, vasculogenesis, and angiogenesis mechanisms. We will delineate more clearly the important links between these observations and those gleaned during the prior highly productive funding period. Specific Focal Points: 1) to exploit the molecular and cellular models of the regulation of the vascular endothelium at the maternal-fetal interface we developed during the initial funding period;and 2) to derive endothelium and trophoblasts from human embryonic stem cells to explore their functional interactions and the hypothesized role of endothelial or other effector cells in differentiation and morphogenesis of the placenta. Project 1: Positive vs. Negative Effects of VEGF on Uterine Artery Endothelial Function during Pregnancy. Project 2: Shear Stress-mediated Pregnancy Adaptations of Uterine Artery Endothelial Cell Functions. Project 3: Human Embryonic Stem Cell-Derived Endothelial Cells. Project 4: A Human Embryonic Stem Cell Model for Trophoblast Differentiation and Placental Morphogenesis. A central role of the embryonic stem cell-derived development of placental morphogenesis (trophoblast and endothelium) that produces effectors, which act to increase placental and uterine blood flows. Concepts of cellular responses to "external mechanosensory input" are a common theme for regulation of trophoblast/endothelial development and of endothelial cell functions. Growth factors like VEG affect uterine vascular functions, including regulating NO vasodilator production by the endothelium in pregnancy by changing cell-cell communication. We believe that physiologic and pathophysiologic exposure of the endothelium to local growth factors like VEGF will change uterine and placental NO production, thereby altering uterine and placental blood flows. A full appreciation of the coordination of the maternal/fetal interface can only be achieved by studying developmental changes and vasodilatation, the latter via NO production and cellular mechanisms for regulating pregnancy specific programmed eNOS activation (e.g. Kinases, cell-cell communication via gap junctions and Ca2+ mobilization). The overall goals and integrated operation of standardized assays and methods in all four Projects of Experimental Cores B (Embryonic Stem Cell) and C (Molecular/Culture). Data from these studies will further our understanding of the basic control of placental and uterine perfusion and mechanisms contributing to fetal pathophysiology in conditions such as pre-eclampsia and IUGR.